Bone is the most transplanted tissue. The gold standard for bone grafts (90% of surgeries) is the autograft. Limitations of this procedure include a limited supply of bone in the body, infection, and pain at the donor site. Therefore, cell-based strategies have received much attention as a replacement therapy, with particular focus on functional tissue engineering. Functional tissue engineering uses physical stimulation to direct cell populations to produce tissue with anatomically and physiologically correct structures and material properties similar to native tissue. Adipose-derived stem cells (ASCs) are a particularly promising cell source for functional tissue engineering applications due to their multilineage differentiation potential and their relative abundance and ease of harvest relative to many other cell lines. However, there is a dearth of information on the mechanobiology of human ASCs (hASCs). Mechanical loads that promote osteodifferentiation of hASCs and creation of functional bone are unknown, as are the mechanotransduction mechanisms associated with load-induced hASC osteodifferentiation. It has been recently shown in bone cells, however, that the primary cilium, a hair-like appendage that exists on almost all cells, plays a crucial mechanotransduction role during bone formation. We have collected unique preliminary data showing that cyclic tensile strain of magnitude 10% accelerates hASC osteodifferentiation and significantly increases calcium accretion. We have also found that hASCs possess a primary cilium, an organelle in bone cells recently shown to play a vital mechanotransduction role during bone formation. Overall Hypothesis: Primary cilia function as sensors of cyclic tensile strain in 3D culture and thereby regulate hASC osteogenesis and functional bone formation. Specific Hypotheses and Objectives/Aims: 1. Primary cilia are responsible, in whole or in part, for transduction of cyclic tensile strain to hASCs in 3D culture. Determine the role of primary cilia in the transduction of cyclic tensile strain to hASCs in 3D culture. Human ASCs will be cultured in 3D collagen gels in both the presence and absence of osteogenic supplements in the culture medium and exposed to 10% cyclic tensile strain - a magnitude we have recently determined to induce and accelerate osteogenesis and increase calcium accretion of hASCs. Primary cilia will be inhibited both biochemically and with a siRNA knockdown strategy and the osteodifferentiation response of hASCs primary cilia characterized. 2. In the absence of primary cilia, bone formed by hASCs will exhibit reduced stiffness and strength. Determine the role of hASC primary cilia in functional bone tissue engineering. Primary cilia will be inhibited as described in SA1. Human ASCs primary cilia will be cultured in 3D collagen gels and exposed to biochemical and mechanical stimuli to induce hASC osteodifferentiation and bone formation. Bone constructs will be tested to failure in tension and compression. Material properties of bone constructs formed by hASCs without primary cilia will be compared to those of bone generated by hASCs with primary cilia and to mature bone. Impact: While it is now understood that mechanical loading is a requirement for successful tissue engineering of load-bearing tissues, it is not known how loading controls stem cell fate or is transduced at the cellular level. Determining the mechanical environment that causes hASC osteodifferentiation and functional bone formation and how those loads are transduced at the cellular level will allow us to optimize hASC response using a synergistic combination of both mechanical and biochemical environments. If it is determined that primary cilia play a vital mechanotransduction role in functional bone formation by hASCs, future work can investigate biochemical or genetic means to induce greater expression of primary cilia in a population of hASCs and/or lengthen hASC primary cilia. Longer primary cilia would be more mechanosensitive (e.g., less load required to bend the primary cilia), allowing for formation of structurally and mechanically robust bone (this is not presently attained by chemical osteogenic supplements alone) by hASCs, even in environments of reduced mechanical load. Such approaches would lead to patient-specific functional bone formation, using a patient's own fat-derived stem cells, and creation of tissue engineered bone capable of withstanding in vivo loads. PUBLIC HEALTH RELEVANCE: The gold standard for bone grafts (90% of surgeries) is the autograft where bone is removed from one portion of the patient's body and grafted to a wound elsewhere. While minimizing tissue rejection, limitations of this procedure include limited supply of bone in the body, infection, and pain at the donor site. Large bone defects can be reconstructed with materials such as metal plates or methyl methacrylate. However, these are foreign materials and may cause a variety of adverse reactions, including irritation, encapsulation of the replacement material, and eventual exposure of the reconstructed area. If this happens, the foreign material has to be removed and the site reconstructed with bone. Bone truly is the best material for reconstruction of bone defects. Being able to create a tissue- engineered bone construct using a patient's own stem cells would eliminate the need for painful bone grafts and/or use of foreign materials to fill critical-sized bone defects.